TasA Matrix YqxM

Fig. 4 Simplified view of the genetic circuitry governing B. subtilis' s lifestyle switch from nomadic to a sedentary existence antagonize SinR activity. Lowered SinR activity results in loss of motility, cell chain formation, and matrix production. The extracellular matrix responsible for proper biofilm development in 3610 consists primarily of an exopolysaccharide (EPS) and a protein, TasA (Branda et al. 2006). Once this matrix is produced, the community develops a high degree of spatiotemporal organization culminating with sporulation occurring preferentially at the tips of aerial structures.

Prior to the discovery of SinR as the master regulator of biofilm formation in B. subtilis, Spo0A and oH were identified as transcriptional factors involved in biofilm development (Branda et al. 2001; Hamon and Lazazzera 2001). Two transcriptional profiling studies had identified members of the Spo0A and oH regulons (Fawcett et al. 2000; Britton et al. 2002). One fifteen-gene operon designated as yveK-T yvfA-F, later renamed epsA-O, under control of both Spo0A and oH, was predicted to encode products likely to be involved in EPS synthesis and export (Branda et al. 2001). EpsA and B are similar to enzymes that regulate EPS chain length, EpsC is similar to nucleotide sugar synthesizing enzymes, EpsD, E, F, H, J, L, and M are all predicted to be glycosyl transferases, EpsK is similar to proteins involved in saccharide export, and EpsG is similar to proteins involved in polymerization of EPS repeating units. Mutants lacking EpsG and EpsH, as well a mutant lacking the entire eps operon, all produce flat colonies and extremely fragile pellicles. Microscopic examination of these mutants revealed that the product(s) of these genes is important for structuring the community. Phase-contrast microscopic analyses made it clear that eps mutants still proliferate as long chains, but these chains no longer align, nor they are bound together (Fig. 5) (Branda et al. 2001). Scanning electron microscopy (SEM) also revealed bare cells with only small amounts of extracellular material remaining.

In addition to the EPS component of the matrix, three proteins, encoded in the three-gene operon yqxM-sipW-tasA, were identified as involved in matrix assembly in a transposon mutant screen for genes involved in biofilm formation (Branda et al. 2004). In-frame deletion mutations in any of the genes of the three-gene operon yqxM-sipW-tasA result in defective pellicle formation and defective colony architecture. Microscopic analyses demonstrate that, like the eps mutants, tasA and yqxM mutants produce cell chains that are not held together and are defective for extracellular matrix production. The tasA and yqxM mutants alone or in combination, as well as a mutant deleted for the entire operon, have similar phenotypes, suggesting that TasA and YqxM act via the same mechanism. The yqxM and tasA genes encode preproteins that are converted to their mature, secreted forms by the product of sipW, a dedicated signal peptidase (Stover and Driks 1999a, 1999b). Previous to these findings, relatively little was known about the function of YqxM and TasA. YqxM was detected in culture supernatants, but only in the presence of high salt, suggesting that it is a cell-surface-associated protein (Stover and Driks 1999a). TasA was detected in the supernatant as well as associated with both cells and spores, and has been reported to have a poorly characterized antimicrobial activity (Serrano et al. 1999; Stover and Driks 1999b).

TasA is present in the biofilm's extracellular matrix. When pellicles were separated from the culture medium, no TasA was detected in the medium (Branda

Fig. 5 Phenotype of eps mutant
Fig. 6 Phenotype of tasA, eps, and tasAeps mutants and extracellular complementation in tasA+eps co-culture

et al. 2006). When mild sonication of the pellicle was used to separate cells from the matrix material, most of the TasA was shown to be present in the matrix fraction. Quite interestingly, TasA remains cell-associated and is not delivered to the matrix fraction when cells lack YqxM, leading to the hypothesis that YqxM is involved in delivering TasA to the matrix (Branda et al. 2006).

While single eps or tasA mutants still produce weak, unstructured pellicles, an eps tasA double mutant produces no pellicle whatsoever, suggesting that the products of these two operons represent the major structural components of the matrix (Fig. 6). Quite strikingly, when an eps mutant is co-cultured with a tasA mutant, there is restoration of the wild pellicle phenotype, suggesting that these components exert their function outside of the cell. In contrast, it was not possible to restore the wild pellicle phenotype by co-culturing tasA and yqxM mutants, consistent with the idea that YqxM is needed to deliver TasA to the matrix. Poly-y-glutamate has also been shown to be an extracellular polymer important for biofilm formation in a different wild strain of B. subtilis (Stanley and Lazazzera 2005). However, mutants unable to produce poly-y-glutamate display a wild type biofilm phenotype in B. subtilis 3610 (Branda et al. 2006).

Mutants lacking SinR or SinI greatly affect biofilm development (Kearns et al. 2005). In the absence of SinI, no pellicle forms and colonies are flat, while the lack of SinR results in extremely wrinkled pellicles and colonies (Fig. 7). eps mutations are epistatic to sinR, i.e., the eps flat colony phenotype is retained in a sinR eps double mutant. DNA footprinting and gel shift analyses using purified SinR revealed that SinR binds directly to the promoter regions of both the eps (Kearns et al. 2005) and yqxM-sipW-tasA operons (Chu et al. 2006). Also, SinR binding to the eps regulatory region is inhibited if purified SinR protein is complexed with purified SinI prior to mixing with DNA (Kearns et al. 2005). Thus, SinR acts as a transcriptional repressor of the genes involved in producing the extracellular matrix, and SinI can antagonize its action.

The involvement of SinR and SinI in the regulation of epsA-O and yqxM-sipW-tasA explains the indirect effects of Spo0A and oH on extracellular matrix synthesis. The sinI and sinR genes are adjacent to each other, with sinI lying upstream. The sinR gene is transcribed primarily from a constitutive promoter dependent on the major housekeeping sigma factor oA, while sinI is transcribed from two oA-dependent promoters, the major one also being dependent on Spo0A~P (Shafikhani et al. 2002). The oH effect is probably due to the fact that spo0A itself contains a oH-dependent promoter (Predich et al. 1992). Therefore, mutants lacking Spo0A or oH will express sinI at a lower level, so that the negative effects of SinR on matrix synthesis will not be antagonized, resulting in defects in biofilm development (Fig. 3). Another regulatory protein known to control B. subtilis biofilm formation is AbrB (Hamon and Lazazzera 2001). However, just exactly how AbrB acts is not yet known.

Spo0A is not the only signal transducer feeding into the pathway regulating extracellular matrix synthesis. Two genes, ylbF and ymcA, when mutated lead to

Wild sin I sinR

Fig. 7 Colony phenotype of sinI and sinR

flat colonies and no pellicles (Branda et al. 2004). In mutants lacking YlbF or YmcA, suppressor mutants take over the surface of the culture and form late-arising pellicles (Kearns et al. 2005). These suppressors that produce hyperwrinkled colonies do, indeed, harbor suppressor mutations in their sinR genes (Kearns et al. 2005). Thus, it appears that YlbF and YmcA function upstream of SinR. Because the expression of ylbF and ymcA does not appear to be regulated by either Spo0A or oH (Britton et al. 2002), we posit that YlbF and YmcA feed into the Sinl-SinR circuitry via a different pathway (Fig. 3).

SinR functions as a master regulator of the lifestyle switch in B. subtilis (Fig. 4). In the model, SinR acts as a direct repressor of the genes involved in extracellular matrix production (epsA-O and yqxM-sipW-tasA). At the same time and through a mechanism that remains largely unknown, SinR acts positively to influence motility and cell separation. During vegetative growth, cells swim, are unit length, and do not produce extracellular matrix. When nutrient limitation is sensed, presumably through both the Spo0A/oH and the YlbF/YmcA pathways, SinI activity increases and SinR is antagonized. In the absence of SinR the expression of matrix components is de-repressed and cell separation and the assembly of motility machinery ceases. As a result, the cells switch to a mode of life where they form chains, become enclosed in a self-produced extracellular matrix, and stop making flagella. Synthesis of the matrix renders the cells able to attain a high degree of spatiotemporal organization, culminating in the production of spores at the tips of aerial projections.

5 The Future of Biofilm Development Research

Elucidation of the genes, proteins, and molecular mechanisms involved in B. subtilis biofilm formation continues and, though much progress has been made in the past 5 years, much remains to be done. Among Gram-positive bacteria, the molecular mechanisms of biofilm formation appear to be species-specific. For example, the master regulators of biofilm formation in B. subtilis (the transcriptional repressor SinR; Kearns et al. 2005), Staphylococcus (the transcriptional activator SarA; Beenken et al. 2003; Valle et al. 2003; Tormo et al. 2005) and Enterococcus (the response-regulator FsrA; Hancock and Perego 2004) are not homologs of each other. In the future, we can expect the combination of genetics, biochemistry, and microscopy to yield an ever-increasing understanding of the molecular mechanisms of biofilm formation unique to many bacteria. Invariably, microbes carry out fascinating, and often unexpected, processes when presented with the greater organizing potential afforded by a surface. Once on a surface, microbial cells can begin long-term relationships with each other; therein lies the transition from unicellularity to multicellularity. Analyses of microbial activities on surfaces will continue to provide new insights into the marvelous and astounding diversity of the microbial world.

Acknowledgements Biofilm work in our laboratory is funded by a grant from the NIH to R.K. (GM58213). K.P.L. was the recipient of an NIH Mentored Clinical Scientist Development Award (K08 AI070561) and A.M.E was the recipient of an NIH postdoctoral fellowship (GM072393).


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